Long lasting effect of new botulinum toxin formulations

ABSTRACT

The invention relates to the use of an animal-protein-free botulinum toxin composition to treat a disease, disorder or condition in a patient in need thereof whereby the animal-protein-free botulinum toxin composition exhibits a longer lasting effect in the patient compared to an animal-protein-containing botulinum toxin composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of Ser. No. 15/336,119,filed Oct. 27, 2016, now U.S. Patent No. 10,143,728, which is aContinuation Application of U.S. patent application Ser. No. 14/567,289,filed Dec. 11, 2014, now U.S. Pat. No. 9,480,731, which claims thebenefit of U.S. Provisional Application Ser. No. 61/915,476, filed Dec.12, 2013, all hereby incorporated entirely by reference.

BACKGROUND OF THE INVENTION

Pioneered in the mid-late 1980s, chemical denervation of the corrugatorand/or procerus muscles with botulinum toxin A (BoNT/A) met many of thecriteria of an ideal cosmetic technique for the treatment of glabellarfrown lines. When carried out by experienced personnel, BoNT/A injectionrapidly and reversibly ameliorates or even eliminates glabellar lines,with virtually no significant adverse effect (Becker-Wegerich P, et al.,Clin Exp Dermatol, 2001 October; 26(7):619-30; Letessier S., J DermatolTreat, 1999; 10(1):31-6 and Alam M, et al., Arch Dermatol, 2002September; 138(9):1180-5).

In 2002, a decade after the first published report on the use of BoNT/Ain the treatment of glabellar frown lines (Carruthers JDA, et al. JDermatol Surg Oncol, 1992 January; 18(1):17-21) the acknowledgedefficacy and tolerability of BoNT/A's effect on glabellar lines wasfinally confirmed in two identical, large, multicenter, placebocontrolled trials (Carruthers J A, et al., J Am Acad Dermatol, 2002June; 46(6):840-9 and Carruthers J, et al., Journal of Plastic andReconstructive Surgery 2003). Since then, BoNT/A has been used widely ina variety of manners to temporarily treat glabellar lines and otherhyperfunctional facial lines, including horizontal forehead lines(‘thinker's wrinkles’) and lateral orbital lines (‘crow's feet’).

Currently commercially available BoNT/A all contain animal proteins suchas albumin. Further commercially available BoNT/A compositions such asBOTOX® have a duration of effect of approximately 3 months for treatingconditions such as crow's feet lines or glabellar lines.

Accordingly, there is a need in the art for a new type of BoNTcomposition that is effective and safe with a duration effect longerthan commercially available BoNT compositions (e.g. BOTOX®, DYSPORT®, orXEOMIN®). The present invention fulfills this need.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 is an image showing the study design and schedule of assessments.

FIG. 2 is an image showing the injection sites. The glabellar linesreceived a single session of either MT10109L or ona-BoN/T. The 0.5 mL(20 U) total injection volume was divided into five symmetricalintramuscular injections: 0.1 mL (4 U) in the procerus muscle, 0.1mL (4U) in each medial corrugator supercilii muscles, and 0.1mL (4 U) in themiddle of each corrugator supercilii muscles.

FIG. 3, comprising FIGS. 3A and 3B, is a series of images showingresponder rate at maximum frown by live assessment (FIG. 3A) PP set and(FIG. 3B) FAS set.

FIG. 4, comprising FIGS. 4A and 4B, is a series of images showing (FIG.4A) responder rate by subject's assessment (PP set) and (FIG. 4B)subject's satisfaction rate (PP set).

FIG. 5 is a graph demonstrating the difference in proportion ofresponders as indicated by investigator assessment rating of glabellarline severity at maximum frown at day 30 for various dosages oflyophilized MT10109 (10U, 20U, and 30U) compared to 20U Botox® (fullanalysis set). Abbreviations. CI=confidence interval; vs =versus. Aresponder was defined as having a glabellar line severity rating of none(0) or mile (1) at the corresponding post-baseline visit. The analysiswas on inputted data using multiple imputation methodology. AMantel-Haenszel chi-square test was used to compare treatments 2 by 2and estimate the differences between the two treatments.

FIG. 6 is a graph depicting the percent of responders as indicated byinvestigator assessment rating of glabellar line severity at all visits(full analysis set). Subjects included in the analysis had to have abaseline glabellar line severity rating of moderate (2) or severe (3). Aresponder was defined as having a severity of none (0) or mild (1) atthe corresponding post-baseline visit.

FIG. 7 is a graph depicting the percent of responders as indicated bysubject's self-assessment of glabellar line improvement (full analysisset). Subjects included in the analysis had to have a baseline glabellarline severity rating of moderate (2) or severe (3). A responder wasdefined as having a score of at least +2 (moderate improvement, i.e.,about 50%) on the 9-point scale.

FIG. 8 is a graph depicting the percent of responders as indicated bysubject's self-assessment of satisfaction with the effect of treatment(full analysis set). Subjects included in the analysis had to have abaseline glabellar line severity rating of moderate (2) or severe (3). Aresponder was defined as having a score of at least 6 (satisfied) on the7-point scale.

FIG. 9 is a set of graphs depicting the results of experiments. The dataincludes percent responding as a function of days post treatment asindicated by investigator assessment at max frown (top left),investigator assessment at rest (middle left), independent reader atfrown (top right), independent reader at rest (middle right), subjectglobal assessment (bottom left), and subject satisfaction (bottomright).

FIG. 10 is a set of graphs depicting the results of experiments. Thedata includes the percent responding as indicated by investigator at maxfrown. The top graph displays the collective data. The graphs at middleleft and bottom left represent the data from site 1, while the graphs atmiddle right and bottom right represent the data from site 2.

DETAILED DESCRIPTION

The invention relates to the use of an animal-protein-free botulinumtoxin composition to treat a disease, disorder or condition in a patientin need thereof whereby the animal-protein-free botulinum toxincomposition exhibits a longer lasting effect in the patient compared toan animal-protein-containing botulinum toxin composition.

In at least one embodiment, the animal-protein-free botulinum toxincomposition is formulated in a liquid form. In certain embodiments theliquid formulation of an animal-protein-free botulinum toxin compositionis the formulation disclosed in US 20100291136, which is incorporatedherein by reference in its entirety. In certain embodiments, the liquidformulation of an animal-protein-free botulinum toxin composition allowsfor the activity of botulinum toxin to be stably maintained under arefrigerated or high temperature condition with the use of neitheranimal-derived protein, such as albumin or gelatin, as a stabilizer forbotulinum toxin nor polar or acidic amino acids such as glutamine,glutamic acid, asparagine or aspartic acid. In at least one embodiment,the animal-protein-free botulinum toxin composition is formulated in alyophilized form. In certain embodiments the lyophilized preparation ofan animal-protein-free botulinum toxin composition is the formulationdisclosed in PCT/KR2012/002418, which is incorporated herein byreference in its entirety. In certain embodiments, the lyophilizedformulation of an animal-protein-free botulinum toxin composition allowsfor maintaining botulinum toxin activity and achieving remarkablysuperior long-term stability even under high-temperature conditionswhich might occur during storage, transportation, or use of botulinumtoxin.

In certain embodiments, the efficacy of the animal-protein-freebotulinum toxin composition (e.g., a liquid formulation or a lyophilizedform) persists longer in the patient compared to ananimal-protein-containing botulinum toxin composition. In certainembodiments, the administration of the animal-protein-free botulinumtoxin composition (e.g., a liquid formulation or a lyophilized form)allows for larger interval time between administrations of the botulinumtoxin composition compared to the interval time when using ananimal-protein-containing botulinum toxin administered at the same orcomparable dose and at the same or comparable sites.

In certain embodiments, the invention provides a treatment regimen thatincludes using an animal-protein-free botulinum toxin compositionwherein the botulinum toxin composition is administered to a patient inneed thereof at a lower dose compared to the dose used with ananimal-protein-containing botulinum toxin composition.

In certain embodiments, the invention provides a treatment regimen thatincludes using an animal-protein-free botulinum toxin compositionwherein the botulinum toxin composition is administered to a patient inneed thereof at a greater time interval between administrations comparedto the time interval used with an animal-protein-containing botulinumtoxin composition (e.g. commercially available botulinum toxin type Aincludes BOTOX®, DYSPORT®, and XEOMIN®; commercially available botulinumtoxin type B includes MyoBloc®).

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About,” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value,as such variations are appropriate to perform the disclosed methods.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

As used herein, the term “animal-protein-free botulinum toxincomposition” refers to a botulinum toxin composition that does notcontain blood derived, blood pooled or other animal derived products(e.g. does not contain albumin). In certain embodiments, theanimal-protein-free botulinum toxin composition is free of human serumalbumin or recombinant human albumin.

As used herein, the term “animal-protein-containing botulinum toxincomposition” refers to a botulinum toxin composition that contains ablood derived, blood pooled or other animal derived product (e.g.contains albumin). In certain embodiments, animal-protein-containingbotulinum toxin composition contains human serum albumin or recombinanthuman albumin.

“Botulinum toxin” means a botulinum neurotoxin as either pure toxin orcomplex, native, recombinant, or modified, and includes botulinum toxintype A, type B, type C₁, type D, type E, type F, and type G. As usedherein, this term excludes non-neurotoxins, such as the cytotoxicbotulinum toxins C₂ and C₃.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound of the invention with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the term “efficacy” refers to the maximal effect(E_(max)) achieved within an assay.

“Local administration” means administration of a pharmaceutical agent toor to the vicinity of a muscle or a subdermal location in a patient by anon-systemic route. Thus, local administration excludes systemic routesof administration, such as intravenous or oral administration.

“Long lasting” or “longer lasting” or “greater duration” refers to thelonger duration of efficacy of an animal-protein-free botulinum toxincomposition when compared to an animal-protein-containing botulinumtoxin composition that is dosed at the same or comparable amount andadministered in the same manner (e.g. by injection) to the same orcomparable location(s).

“Peripheral administration” means administration to a location away froma symptomatic location, as opposed to a local administration.

“Pharmaceutically acceptable” refers to those properties and/orsubstances which are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability. “Pharmaceutically acceptable carrier” refers to amedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s) and is not toxic to the host towhich it is administered.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs or symptoms of pathology, for the purpose of diminishingor eliminating those signs or symptoms.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the invention (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has a condition contemplatedherein, a symptom of a condition contemplated herein or the potential todevelop a condition contemplated herein, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect acondition contemplated herein, the symptoms of a condition contemplatedherein or the potential to develop a condition contemplated herein. Suchtreatments may be specifically tailored or modified, based on knowledgeobtained from the field of pharmacogenomics.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention is based on the discovery that ananimal-protein-free botulinum toxin composition exhibited an improvedoutcome in a recipient patient compared to an otherwise identicalpatient receiving an animal-protein-containing botulinum toxincomposition when the patient was evaluated 16 weeks after theadministration of the botulinum toxin composition. That is, theanimal-protein-free botulinum toxin composition exhibits a longerlasting effectiveness compared to an animal-protein-containing botulinumtoxin composition.

Liquid Animal-Protein-Free Formulation

Compositions useful in the invention include an animal-protein-freebotulinum toxin composition. In certain embodiments, theanimal-protein-free botulinum toxin composition is in a liquidformulation.

In certain embodiments, the liquid pharmaceutical composition used inthe present invention comprises botulinum toxin, polysorbate 20, andmethionine.

In certain embodiments, the liquid pharmaceutical composition used inthe present invention comprises botulinum toxin, polysorbate 20,methionine and isoleucine.

In certain embodiments, the liquid pharmaceutical composition comprisesa botulinum toxin, polysorbate 20 and methionine and optionallyisoleucine.

With the employment of polysorbate 20, methionine and optionallyisoleucine, instead of an animal-derived protein such as albumin orgelatin, as stabilizers for botulinum toxin, the liquid pharmaceuticalcomposition used in the present invention excludes the potential risk ofinfecting the recipient with serum-derived pathogens or microorganismsand is thus safe for ingestion into the body. In addition, the use ofthe stabilizers polysorbate 20, methionine and optionally isoleucineallows for higher stability to botulinum toxin composition at around 25°C. to about 37° C. Thus, in terms of the storage stability of botulinumtoxin composition at 25° C. to about 37° C., the liquid pharmaceuticalcomposition is very useful for storing botulinum toxin composition underan emergency condition such as an environment without maintaining lowtemperature, thus being superior to conventional liquid pharmaceuticalcompositions employing either detergents or amino acids.

In certain embodiments, the methionine is present in an amount of about0.5 to 100 μmol per 100 units of botulinum toxin, and preferably rangesin concentration from about 0.5 to 100 mM and more preferably from about25 to about 75 mM. In another embodiment, the methionine ranges inconcentration from about 0.5 mM to about 100 mM.

A methionine content less than 0.5 μmol per 100 units of botulinum toxincannot guarantee the stabilization of the botulinum toxin to a desirablelevel upon long-term storage at room temperature. On the other hand,when methionine is used in an amount exceeding 100 μmol per 100 units ofbotulinum toxin, the excess increment may not promise an additionalstabilization effect in addition to incurring an economic disadvantage.In the liquid pharmaceutical composition used in the present invention,methionine properly ranges in concentration from 0.5 to 100 mM when thebotulinum toxin has a concentration of 100 units/mL. Its properconcentration is adjusted to about 25 mM to about 75 mM in considerationof the concentration range of polysorbate 20. When the concentration ofmethionine is below 25 mM in the liquid pharmaceutical composition usedin the present invention, its long-term stabilization effect onbotulinum toxin at room temperature does not reach the desirable level,which is obtainable in the proper concentration range of botulinumtoxin. On the other hand, a methionine concentration exceeding 75 mMdoes not provide any additional effect. In certain embodiments,polysorbate 20 is present in an amount of 0.01 to 50 mg per 100 units ofbotulinum toxin and preferably ranges in concentration from 0.01 to 50mg/mL and more preferably form 0.1 to 2.5 mg/mL.

Polysorbates are a class of emulsifiers used in some pharmaceuticals andin food preparation. They are often used in cosmetics to dissolveessential oils into water-based (oil-in-water) products. There are manykinds of polysorbates that are classified by a number referring to thetotal number of oxyethylene groups, such as polysorbate 20, 40, 60 and80. In certain embodiments, the liquid pharmaceutical composition usedin the present invention employs polysorbate 20 (commercially availableas brand name Tween 20) as a stabilizer for botulinum toxin.

If the liquid pharmaceutical composition used in certain embodiments ofthe present invention contains polysorbate 20 in an amount less than0.01 mg per 100 units of botulinum toxin, its long-term stabilizationeffect on botulinum toxin at room temperature does not reach a desirablelevel. On the other hand, a polysorbate 20 concentration exceeding 50mg/mL does not provide any additional effect in addition to incurring aneconomic disadvantage. At a concentration of 100 units/mL of botulinumtoxin in the liquid pharmaceutical composition used in certainembodiments of the present invention, polysorbate 20 is properly presentin an amount of about 0.01 mg/mL to about 50 mg/mL and preferably in anamount of about 0.1 mg/mL to about 2.5 mg/mL when the methionineconcentration is taken into consideration. When the concentration ofpolysorbate 20 in the liquid pharmaceutical composition used in certainembodiments of the present invention is less than 0.1 mg/mL, itslong-term stabilization effect on botulinum toxin at room temperaturedoes not reach a desired level, which is obtainable by the targetconcentration of polysorbate 20. On the other hand, a polysorbate 20concentration exceeding 2.5 mg/mL does not provide any additionaleffect.

The botulinum toxin, a constituent of the liquid pharmaceuticalcomposition used in certain embodiments of the present invention, may beone selected from among serotypes A, B, C, D, E, F and G. The termbotulinum toxin is a generic term embracing the family of toxinsproduced by the anaerobic bacterium Clostridium botulinum and, to date,seven immunologically distinct neurotoxins serotypes have beenidentified. These have been given the designations A, B, C, D, E, F andG, which differ one from the other in their effects on target animals,and paralysis extent and duration. All serotypes of botulinum toxin areknown to act as a neurotoxin by inhibiting the neurotransmitteracetylcholine at neuromuscular junctions.

The botulinum toxin of the liquid pharmaceutical composition used incertain embodiments of the present invention may be in a non-complexform or in a complex form with another protein. Botulinum toxin serotypeA, B, C, D, E, F or G alone, synthesized by Clostridium botulinum,itself has a molecular weight of approximately 150 kDa. When expressedin Clostridium botulinum, the botulinum toxin forms various complexeswith hemagglutinin proteins and non-hemagglutinin proteins which aid andprotect the activity thereof. Naturally occurring botulinum type Acomplexes have a molecular weight of approximately 900 kDa, 500 kDa or300 kDa. Molecular weights are measured to be approximately 500 kDa forbotulinum toxin type B complexes and type C complexes, approximately 300kDa or 500 kDa for type D complexes, and approximately 300 kDa for typeE and type F complexes.

In certain embodiments, the concentration of the botulinum toxin in theliquid pharmaceutical composition preferably ranges from 50 to 5,000units/mL depending on the general use thereof.

In certain embodiments, the liquid pharmaceutical composition using inthe present invention has a pH of about 5.5 to 7.0. In certainembodiments, when the liquid pharmaceutical composition used in thepresent invention is adjusted to a pH of about 5.5 to about 7.0,botulinum toxin is stably maintained at room temperature (particularly40° C.) for a long period of time.

The liquid pharmaceutical composition can be readily prepared because itemploys a detergent and an amino acid(s) without a lyophilizationprocess.

Lyophilized Animal-Protein-Free Botulinum Toxin Formulation

Compositions useful in the invention include an animal-protein-freebotulinum toxin composition. In certain embodiments, theanimal-protein-free botulinum toxin composition is a lyophilizedpreparation of botulinum toxin. For example, the lyophilized preparationof botulinum toxin composition useful in the invention does not containa protein stabilizer derived from an animal.

In certain embodiments, the present invention provides a lyophilizedpreparation of a botulinum toxin composition comprising: 1) botulinumtoxin; 2) polysorbate; 3) methionine; and 4) one or more componentsselected from a group consisting of sugar, sugar alcohol, an ioniccompound, and a combination thereof.

In certain embodiments, when the botulinum toxin composition is formedas a lyophilized preparation, the component(s) play(s) a role inmaintaining the activity of the botulinum toxin composition whilefacilitating stabilization at temperatures greater than roomtemperature. At the time of lyophilization, preparations containing 1)botulinum toxin; 2) polysorbate; and 3) methionine exhibit reducedstability, and when they are formulated as a liquid preparation theyexperience reduced stability at temperatures greater than roomtemperature; however, the lyophilized preparation of botulinum toxincomposition used in certain embodiments of the present invention notonly maintains the activity of the botulinum toxin composition attemperatures greater than room temperature, but also is excellentrelative to storage stability over long periods.

The botulinum toxin may be derived from Clostridium botulinum. Thebotulinum toxin may be separated and refined from these strains by knownmethods, or else a commercially available product may be used.

The botulinum toxin may be randomly selected from a group consisting ofbotulinum Serotypes A, B, C, D, E F and G.

The botulinum toxin present in the lyophilized preparation may be informs containing and not containing proteins in complexes. The activityof the botulinum toxin is unaffected whether or not the protein is in acomplex. In the lyophilized preparation of botulinum toxin compositionused in certain embodiments of the present invention, polysorbate, whichis one of the stabilizers of the botulinum toxin, is a non-ionicsurfactant, and is primarily used as an emulsifier in the pharmaceuticalor food industries. As polysorbate types, there are polysorbate 20, 40,60, 80 or 100, based on the total number of oxyethylene groups. For thelyophilized preparation of botulinum toxin composition used in certainembodiments of the present invention, it is acceptable to use any fromamong these. The polysorbate may be present at an amount of about 0.01to about 2 mg per 100 units of the botulinum toxin. If polysorbate ispresent within this range, then the activity of the botulinum toxin canbe maintained even at temperatures greater than room temperature, andlong-term storage stability can be maintained.

In certain embodiments, methionine, which is one of the stabilizers, mayalso be used as a substitute for animal proteins such as albumin orgelatin, as the stabilizer for the botulinum toxin. The methionine maybe present at an amount of about 0.01 to 10 mg per 100 units of thebotulinum toxin. If methionine is comprised within this range, then theactivity of the botulinum toxin composition can be maintained even attemperatures greater than room temperature, and long-term storagestability can be maintained.

Different from existing liquid preparations, the lyophilized preparationof botulinum toxin composition used in certain embodiments of thepresent invention further comprises at least one from among sugar, sugaralcohol or an ionic compound, as an additional component aside frommethionine and polysorbate. Sugar is known to protect the denaturationof the macromolecule. As a sugar that may be used in the lyophilizedpreparation used in certain embodiments of the present invention,trehalose, sucrose, maltose, fructose, lapinose, lactose or glucose maybe used; however, the use thereof is not limited to these types. Thesugar may be at an amount of about 0.1 to 50 mg per 100 units of thebotulinum toxin. If sugar is present within this range, then theactivity of the botulinum toxin composition can be maintained even attemperatures greater than room temperature, and long-term storagestability can be maintained.

Sugar alcohol is known to stabilize the macromolecule underlyophilization conditions, and to usefully prevent denaturation. As asugar alcohol that may be used in the lyophilized preparation used incertain embodiments of the present invention, cyclodextrin, mannitol,sorbitol, glycerol, xylitol or inositol, etc. may be used. The sugaralcohol may be at an amount of about 0.1 to 50 mg per 100 units of thebotulinum toxin. If sugar alcohol is present within this range, then theactivity of the botulinum toxin composition can be maintained even attemperatures greater than room temperature, and long-term storagestability can be maintained.

In certain embodiments, “ionic compound” refers to a salt, buffer, etc.The ionic compound works with the macromolecule through specific ornon-specific binding. Salt can increase thermal stability, increasesolubility and decrease the degree of aggregation. However, caution isrequired with high concentrations of salt due to the observed tendencyfor protein denaturation. As the ionic compound, sodium chloride, sodiumphosphate, ammonium phosphate, magnesium sulfate, sodium acetate, sodiumlactate, sodium succinate, sodium propionate or potassium phosphate maybe used; however, the use thereof is not limited to these types. Theionic compound may be present at an amount of about 0.1 to 10 mg per 100units of the botulinum toxin. If the ionic compound is present withinthis range, then the activity of the botulinum toxin composition can bemaintained even at temperatures greater than room temperature, andlong-term storage stability can be maintained.

In certain embodiments, the lyophilized preparation of botulinum toxincomposition used in the present invention is manufactured from theculture of Clostridium botulinum that has been cultured in a specificmedium, although this is not limited. The botulinum toxin complex ispurified through a series of acid precipitations as a crystallinecomplex composed of active high molecular weight toxin protein andrelated

Hemagglutinin protein. The crystalline complex is dissolved in asolution containing salt and stabilizer, and the lyophilized preparationof botulinum toxin composition is produced by undergoing a freeze dryingprocess.

Method

The invention relates to the use of an animal-protein-free botulinumtoxin composition to treat a disease, disorder or condition in a patientin need thereof whereby the animal-protein-free botulinum toxincomposition exhibits a longer lasting effect in the patient compared toan animal-protein-containing botulinum toxin composition. For example,it was observed that an animal-protein-free botulinum toxin compositionexhibited a greater improvement on the therapeutic outcome in a patientcompared to the outcome of the patient receiving ananimal-protein-containing botulinum toxin composition.

In certain embodiments, the efficacy of the animal-protein-freebotulinum toxin composition (e.g., a liquid formulation or a lyophilizedform) persists longer in the patient compared to ananimal-protein-containing botulinum toxin composition. In certainembodiments, the administration of the animal-protein-free botulinumtoxin composition (e.g., a liquid formulation or a lyophilized form)allows for larger interval time between administrations of the botulinumtoxin composition compared to the interval time when using ananimal-protein-containing botulinum toxin administered at the same orcomparable dose and at the same or comparable sites.

In certain embodiments, the invention provides a treatment regimen thatincludes using an animal-protein-free botulinum toxin compositionwherein the botulinum toxin composition is administered to a patient inneed thereof at a lower dose compared to the dose used with ananimal-protein-containing botulinum toxin composition.

In certain embodiments, the invention provides a method for treating acondition in a patient in need thereof, the method comprising the stepof locally administering a therapeutically effective amount of ananimal-protein-free botulinum toxin composition, whereby at least onesymptom of the condition is thereby effectively alleviated for a periodof time longer than that of an animal-protein-containing botulinum toxincomposition.

In certain embodiments, the invention provides a treatment regimen thatincludes using an animal-protein-free botulinum toxin compositionwherein the botulinum toxin composition is administered to a patient inneed thereof at a greater time interval between administrations comparedto the time interval used with an animal-protein-containing botulinumtoxin composition.

In certain embodiments, the invention provides a method for treating acondition in a patient in need thereof, the method comprising the stepof locally administering a therapeutically effective amount of ananimal-protein-free botulinum toxin composition, wherein the compositionis administered at an interval of time between a first treatment and asecond treatment effective to maintain alleviation of at least onesymptom of the condition, that is greater than the interval of time foran animal-protein-containing botulinum toxin composition dosed at thesame or comparable amount and administered in the same manner (e.g. byinjection) to the same locations as that of the animal-protein-freecomposition.

In certain embodiments, the time between the first treatment and secondtreatment of a condition in a human patient using an animal-protein-freebotulinum toxin composition is at least one month, at least 2 months, atleast 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks,at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least23 weeks, at least 24 weeks, at least 25 weeks, at least 26 weeks, atleast 27 weeks, at least 28 weeks, at least 29 weeks, at least 30 weeks,at least 31 weeks, at least 32 weeks, at least 33 weeks, at least 34weeks, at least 35 weeks, at least 36 weeks, at least 37 weeks, at least38 weeks, at least 39 weeks, at least 40 weeks, at least 41 weeks, atleast 42 weeks, at least 43 weeks, at least 44 weeks, at least 45 weeks,at least 46 weeks, at least 47 weeks, at least 48 weeks, at least 49weeks, at least 50 weeks, at least 51 weeks, at least 52 weeks or more.In certain embodiments, the time between the first treatment and secondtreatment using an animal-protein-free botulinum toxin composition isgreater than 3 months for the effective alleviation of at least onesymptom of a condition in a patient. In certain embodiments, the timebetween the first treatment and second treatment using ananimal-protein-free botulinum toxin composition of the present inventionis greater than 16 weeks for the effective alleviation of at least onesymptom of a condition in a patient.

In certain embodiments, the animal-protein-free botulinum toxincomposition of the present invention can be used for cosmetic purposessuch as to treat skin contour deficiencies, including wrinkles, of anindividual.

In certain embodiments, the animal-protein-free botulinum toxincomposition of the present invention can be used to treat glabellarlines.

In certain embodiments, the animal-protein-free botulinum toxincomposition of the present invention can be used to treat lateralcanthal lines.

In certain embodiments, the animal-protein-free botulinum toxincomposition of the present invention can be used for non-cosmeticpurposes. In certain embodiments, the animal-protein-free botulinumtoxin composition of the present invention can be used to treat variousdiseases and disorders including but not limited to detrusoroveractivity associated with a neurologic condition, chronic migraine,upper limb spasticity, cervical dystonia, primary axillaryhyperhidrosis, blepharospasm and strabismus, idiopathic overactivebladder, and the like.

In certain embodiments, the animal-protein-free botulinum toxincomposition of the present invention can be used for non-cosmeticpurposes. Non-limiting examples of a non-cosmetic purpose includes butis not limited to the treatment of urinary incontinence due to detrusoroveractivity associated with a neurologic condition [e.g., spinal cordinjury (SCI), multiple sclerosis (MS)] in adults who have an inadequateresponse to or are intolerant of an anticholinergic medication,prophylaxis of headaches in patients with chronic migraine (e.g., ≥15days per month with headache lasting 4 hours a day or longer), treatmentof upper limb spasticity in adult patients, treatment of cervicaldystonia in adult patients, to reduce the severity of abnormal headposition and neck pain, treatment of severe axillary hyperhidrosis thatis inadequately managed by topical agents in adult patients, treatmentof blepharospasm associated with dystonia, treatment of strabismus inpatients ≥12 years of age.

However, the invention should not be limited to only the diseases,disorder, or conditions disclosed herein. Rather, the inventionencompasses the use of the animal-protein-free botulinum toxincomposition of the present invention to treat any disease for wherebotulinum toxin has been used. For example, botulinum toxin has beenproposed for or has been used to treat otitis media of the ear (U.S.Pat. No. 5,766,605), inner ear disorders (U.S. Pat. Nos. 6,265,379;6,358,926), tension headache, (U.S. Pat. No. 6,458,365), migraineheadache pain (U.S. Pat. No. 5,714,468), post-operative pain andvisceral pain (U.S. Pat. No. 6,464,986), hair growth and hair retention(U.S. Pat. No. 6,299,893), psoriasis and dermatitis (U.S. Pat. No.5,670,484), injured muscles (U.S. Pat. No. 6,423,319) various cancers(U.S. Pat. Nos. 6,139,845), smooth muscle disorders (U.S. Pat. No.5,437,291), and neurogenic inflammation (U.S. Pat. No. 6,063,768). Inaddition, botulinum toxins have been used in clinical settings forcosmetic applications as well as the treatment of neuromusculardisorders characterized by hyperactive skeletal muscles. Botulinum toxinis currently used in the treatment of hyperhidrosis, and can also beused in the treatment of achalasia, chronic focal neuropathies, analfissure, vaginismus, spastic disorders associated with injury or diseaseof the central nervous system (including, for example, trauma, stroke,multiple sclerosis, Parkinson's disease, cerebral palsy, and the like),focal dystonias affecting the limbs, face, jaw, or vocal cords,temporomandibular joint disorder (TMJ), diabetic neuropathy, woundhealing disorders, excessive salivation, vocal cord dysfunction (VCD)including spasmodic dysphonia, and tremor. Botulinum toxin type A hasbeen approved by the U.S. Food and Drug Administration for the treatmentof blepharospasm, strabismus, hemifacial spasm, cervical dystonia,migraine headaches, overactive bladder and detrusor overactivityassociated with a neurological condition.

In certain embodiments, methods of the invention can be useful for thetreatment, reduction of symptoms, and/or prevention of achalasia, analfissure, anismus, blepharospasm, cerebral palsy, cervical dystonia,cervicogenic headache, hemifacial spasm, dyshidrotic eczema, dysphagia,dysphonia, esophageal dysmotility, esophageal muscular ring, esotropia(infantile), eyelift, facial myokymia, gait disturbances (idiopathictoe-walking), generalized dystonia, hemifacial spasm, hyperfunctionalfacial lines (glabellar, forehead, crows' feet, down-turned angles ofthe mouth), hyperhidrosis, incontinence (spinal cord injury), migraineheadache, myoclonus, myofascial pain syndrome, obstructive urinarysymptoms, pancreas divisum pancreatitis, Parkinson's disease,puborectalis syndrome, reduction of surgical scar tension, salivaryhypersecretion, sialocele, sixth nerve palsy, spasticity, speech/voicedisorders, strabismus, surgery adjunct (ophthalmic), tardive dyskinesia,temporomandibular joint disorders, tension headache, thoracic outletsyndrome, torsion dystonia, torticollis, Tourette's syndrome, tremor,whiplash-associated neck pain, pain, itching, inflammation, allergy,cancer and benign tumors, fever, obesity, infectious diseases, viral andbacterial, hypertension, cardiac arrhythmias, vasospasm,atherosclerosis, endothelial hyperplasia, venous thrombosis, varicoseveins, aphthous stomatitis, hypersalivation, temporomandibular jointsyndrome, sweating, body odor, acne, rosacea, hyperpigmentation,hypertrophic scars, keloid, calluses and corns, skin wrinkling,excessive sebum production, psoriasis, dermatitis, allergic rhinitis,nasal congestion, post nasal drip, sneezing, ear wax, serous andsuppurative otitis media, tonsil and adenoid hypertrophy, tinnitus,dizziness, vertigo, hoarseness, cough, sleep apnea, snoring, glaucoma,conjunctivitis, uveitis, strabismus, Grave's disease, asthma,bronchitis, emphysema, mucus production, pleuritis, coagulationdisorders, myeloproliferative disorders, disorders involvingeosinophils, neutrophils, macrophages and lymphocytes, immune toleranceand transplantation, autoimmune disorders, dysphagia, acid reflux,hiatal hernia, gastritis and hyperacidity, diarrhea and constipation,hemorrhoids, urinary incontinence, prostatic hypertrophy, erectiledysfunction, priapism and Peyronie's disease, epididymitis,contraception, menstrual cramps, preventing premature delivery,endometriosis and fibroids, arthritis, osteoarthritis, rheumatoid,bursitis, tendonitis, tenosynovitis, fibromyalgia, seizure disorders,cerebral palsy, spasticity, headache, depression and neuralgias.

In certain embodiments, the treatment regimen of the invention compriseslocal administration of the animal-protein-free botulinum toxincomposition. In certain embodiments, the treatment regimen comprisesparenteral administration of the animal-protein-free botulinum toxincomposition. In certain embodiments the animal-protein-free compositionis administered by injection, topically or by implantation of acontrolled release implant. Examples of administration by injectioninclude intramuscular injection, non-intramuscular injection,intra-articular injection, extra-articular injection, peri-articularinjection, or subcutaneous injection.

In certain embodiments, the treatment regimen of the invention isbeneficial due to the longer lasting effect and improved outcomesassociated with using animal-protein-free botulinum toxin compositionswhen compared to using animal-protein-containing botulinum toxincompositions. Without wishing to be bound by any particular theory, itis believed that in certain embodiments the long lasting effect of theanimal-protein-free botulinum toxin composition allows for a longerinterval time between treatments with the animal-protein-free botulinumtoxin composition compared to the interval time when using ananimal-protein-containing botulinum toxin composition.

Furthermore, without wishing to be bound by any particular theory, it isbelieved that in certain embodiments the unique formulation of thecompositions using in the invention allows for the longer lasting effectof the composition in the patient.

In certain embodiments, by increasing the interval time betweentreatments, the treatment regimen allows for a reduction in thefrequency of treatment. Therefore, the invention provides the potentialfor enhanced patient convenience. The increased interval time betweentreatment and lower frequency of treatment also has the potential toprovide reduced side effects.

In certain embodiments, the treatment regimen of the invention isbeneficial due to the long lasting effect and improved outcomesassociated with using the liquid animal-protein-free botulinum toxincomposition or the lyophilized animal-protein-free botulinum compositionas described herein.

In certain embodiments, in a treatment method of the invention, atherapeutically effective level botulinum toxin is present in therecipient patient for an extended period of time of at least 16 weeks,at least 18 weeks, at least 20 weeks, at least 22 weeks, at least 24weeks, at least 26 weeks, at least 28 weeks, at least 30 weeks, at least32 weeks, at least 34 weeks, at least 36 weeks, at least 38 weeks, atleast 40 weeks, at least 42 weeks, at least 44 weeks, at least 46 weeks,at least 48 weeks, at least 50 weeks, or more.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 The Efficacy and Safety of Liquid Type Botulinum Toxin Type Afor the Management of Moderate to Severe Glabellar Frown Lines: AParallel, Randomized, Double-Blind, Multi-Center, Active DrugControlled, Phase III Clinical Trial

Botulinum toxin A (BoNT/A) has been used widely in a variety of mannersto correct unwanted glabellar lines and other hyperfunctional faciallines. However, most of currently using BoNT/A requires dilution withsaline solution, which is very inconvenient for the user, and usually ishard to make the exact concentration every time. The results presentedherein is based on experiments conducted that compared the efficacy andsafety of newly developed liquid type BoNT/A (MT10109L) andonabotulinumtoxinA (BOTOX®; ona-BoNT/A) for moderate to severe glabellarlines.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

This study was a prospective, randomized, double-blind, parallel, activedrug controlled, Phase III clinical trial for the evaluation of theefficacy and safety of MT10109L on the glabellar frown line correctionwhich was performed in three centers (St. Paul's hospital, Catholicuniversity of Korea; Inha university hospital; Kyunghee universityhospital at Gangdong) in South Korea. The study and all appropriateamendments were reviewed and approved by the institutional review boardat each participating center in accordance with the guidelines publishedin the Declaration of Helsinki (South Africa, 1996 amendment) and GoodClinical Practice guideline. All participants gave written informedconsent to take part in the study.

Participants

Male and female volunteers aged 20-65 years with glabellar lines werescreened by investigators. According to Facial Wrinkle Scale (FWS),subjects with moderate to severe (severity score 2 to 3) glabellar frownlines were enrolled in this study (Table 1). Exclusion criteria includedany medical condition (e.g. myasthenia gravis, Lambert-Eaton syndrome,amyotrophic lateral sclerosis) that may place the patient at risk withbotulinum toxin, prior use of medications that may affect theneuromuscular junction (e.g., muscle relaxants, spectinomycin HCl,aminoglycosides, polypeptide antibiotics, anticholinergics,benzodiazepines) or any allergies or hypersensitivity to theinvestigational drugs or their components. Other exclusion criteriaincluded previous treatment with botulinum toxin within 3 months, otherprocedures that may have affected glabellar and forehead lines within 6months, any history of glabellar treatment (including forehead) such asface lifting and/or permanent implants, or scars that may affect thetreatment results. Patients whose glabellar lines that could not besatisfactorily improved even with manual stretching were also excluded.Patients were not eligible if they had dermatological disorders orinfection at potential injection sites, or a history of facial nerveparalysis or ptosis. Pregnant or lactating women were excluded.

TABLE 1 Scales used to assess the effectiveness of MT10109L andOna-BoNT/A Measure scale Description Facial Wrinkle 3 Severe; linesappear clearly formed. Scale, The bottoms of the deepest lines aremaximal frown 2 Moderate; lines appear clearly formed. The bottoms ofthe deepest lines 1 Mild; lines noted 0 None; lines not noted FacialWrinkle 3 Severe; lines readily apparent Scare, rest 2 Moderate; linesnoticeable 1 Mild; lines somewhat noticeable 0 None; lines notnoticeable

Study Design

All eligible subjects were randomized into two groups at a 1:1 ratio andfollowed a 16 weeks duration study design (FIG. 1.). At Visit 2 (0 week,baseline), each subject received a 5 point intramuscular injection witha total dose of 20U (4 U/0.1 ml) of liquid BoNT/A (MT10109L; MedytoxInc., Cheongwon-gun, Korea) or onabotulinumtoxinA (BOTOX®; ona-BoNT/A)in a double blind manner. The 0.5 mL total injection volume was dividedinto five injections: 0.1 mL (4 U) in the procerus muscle, 0.1 mL (4 U)in each medial corrugator supercilii muscles, and 0.1 mL (4 U) in themiddle of each corrugator supercilii muscles (FIG. 2). For this clinicalstudy, MT10109L (0.625 ml/vial (4 U/0.1 ml)) and 50 U ona-BoNT/A wasused. MT10109L did not require additional dilution for its liquidnature, but ona-BoNT/A was dissolved in 1.25 ml of 0.9% NaCl 1.25 ml tomake it 4 U per 0.1 ml.

Efficacy Measures

During the observation period of 16 weeks, the subjects were assessed at4, 10, 16 weeks. At each visit, both the investigator and the patientassessed efficacy and safety. In addition, standardized digitalphotographs of the treated facial area were taken in the same settingusing the same equipment (EOS 600d, Canon Inc., Tokyo, Japan) to ensurereproducibility (FIG. 1). Physicians assessed the glabellar lineseverity at maximum frown and at rest using FWS by live assessment. Allinvestigators in each center were provided the standardized photographgrading system. Three blinded raters assessed the photographs at maximumfrown and at rest according to the FWS (Table 1).

The primary efficacy end point was the percentage of responders atmaximum frown at week 4 based on the investigator's live assessment(face-to-face observation). Secondary efficacy end point included: 1)percentage of responders at maximum frown at weeks 16; 2) percentage ofresponders of glabellar lines at rest based on investigator's liveassessment at weeks 4 and 16; and 3) percentage of responders at maximumfrown and at rest based on photographic assessment at weeks 4. Inaccordance with previous studies for ona-BoNT/A, responders were definedas those who have post-treatment FWS scores of 0 or 1 with pretreatmentFWS scores of 2 or 3. This means an improvement of at least 1 point forthe subjects with moderate wrinkles and at least 2 points for thesubjects with severe wrinkles. In addition, the glabellar lineimprovement rates determined by the subject's own assessment andsatisfaction rates at weeks 4, 10 and 16 as secondary efficacy end pointwere also included. Subjects assessed the change of the line severityusing 9 point scale from +4 (100% improvement) to 0 (no change) to −4(100% worse), and rated their degree of satisfaction with the treatmentin a 7 point scale from −3 (very unsatisfied) to +3 (very satisfied).Scores of more than +2 points (moderately improved) was considered asimprovement. Moreover, scores of more than 6 points (satisfied) wereconsidered as satisfaction.

Safety Measures

During the study, physical examination and vital sign were checked everyvisit.

Investigator- and subject-reported signs and symptoms, and laboratorytest (Complete Blood Count, and Blood Chemistry) were performed atscreening day and weeks 16. Urine-hCG was checked at screening,treatment day, and week 16. Summary and analysis of adverse events wasperformed about all adverse events occurred after receipt of consent.Incidence of adverse events were documented by comparing incidence ofall adverse events, incidence of adverse events related to study drug,and incidence of severe adverse events.

Statistical Methods

All subjects with data for primary end points were included in the fullanalysis set (FAS). The per protocol (PP) set was the subset of subjectsof the FAS who did not commit any major protocol violation. For theprimary efficacy end point parameter, the lower limit of 97.5% one-sidedconfidential interval (CI) for the difference in responder rates betweentwo groups was calculated. The interpretation of the CI was based on thenull hypothesis that the expected difference in responder rates betweenthe treatment groups was lower than the non-inferiority margin of −15%.If the lower bound of the estimated CI exceeded the limit of −15%, onecould conclude that the MT10109L was not inferior to ona-BoNT/A. Thisconfirmatory analysis was based on the PP analysis. For secondaryefficacy end point, paired t-test, Pearson's chi-square test, orFisher's exact test were performed.

Fisher's exact test was performed to test for between-group differencesin adverse events. For laboratory variables, blood pressure, and heartrate, the Wilcoxon signed-rank test was performed for within-groupanalyses, and the Wilcoxon rank-sum test was used for between groupanalyses of data at exit.

The results of the experiments are now described.

Baseline Demographic Characteristics

Of 168 subjects enrolled, 159 subjects completed the study and thereforeconstituted the PP set: 78 subjects in MT10109L group and 81 subjects inona-BoNT/A group. The enrolled subject's age was from 20 to 65, and themean age was 48.94 year old in MT10109L group and 49.86 year old inona-BoNT/A group, respectively. All subjects were Koreans. Demographicsof the two groups were comparable, and two groups did not differ intheir pretreatment line severity either at rest or maximum frown beforetreatment. All of subjects had moderate to severe glabellar frown linesat rest and the majority of subjects had severe glabellar frown lines atmaximum frown (56.41% for MT10109L group; 50.62% for ona-BoNT/A group)(Table 2).

TABLE 2 Subject Demographic Characteristics (N = 159, PP set) MT10109LOna-BONT/A N = 78 N = 81 n (%) n (%) p-value Age n 78 81 Mean ± SD 48.94± 9.13  49.86 ± 9.13  0.5226* 20~29 3(3.85) 3(3.70) 0.8081† 30~397(8.97)  9(11.11) 40~49 28(35.90) 22(27.16) 50~59 32(41.03) 39(48.15)60~65  8(10.26) 8(9.88) Gender n 78 81 male 19(24.36) 15(18.52) 0.3692†female 59(57.64) 66(81.48) Botulinumtoxin injections history n 78 81 yes15(19.23) 12(14.81) 0.4585† no 63(80.77) 69(85.19) No. of Botulinumtoxininjection n 15 12 Mean ± SD 1.93 ± 0.26 2.00 ± 0.43 0.6547+ Elapsed timeafter the administration n 15 12 of botulinumtoxin injections (month)Mean ± SD 22.18 ± 13.28 20.31 ± 16.44 0.6428+ at screening, n 78 81investigator's live assessment 3(severe) 44(56.41) 41(50.62) 0.4641† atmaximum frown (point) 2(moderate) 34(43.59) 40(49.38) *two sample t-test+Wilcoxon rank sum test †Pearson's chi-square test

Investigator's Assessment

Both groups showed significant improvement of glabellar lines. Fourweeks after injection, percentage of responders at maximum frown by liveassessment for the PP set was 87.18% in MT10109L group and 87.65% inona-BoNT/A group. In addition, percentage of responders of FAS inMT10109L group and ona-BoNT/A group was similar to that of PP set, being85.54% and 85.71%, respectively (FIG. 3A). The 97.5%

CIs for the difference in percentage of responders between the twotreatment groups (−10.79% for PP>−15%; −10.47% for FAS>−15%)demonstrates that MT10109L was not inferior to ona-BoNT/A.

The percentage of responders at maximum frown by live assessment atweeks 16 was significantly lower in ona-BoNT/A group than MT10109Lgroup. The percentage of responders in PP set was 62.34% in MT10109Lgroup and 40.51% in ona-BoNT/A group (p value=0.0064) (Table 3). And thepercentage of responders in FAS set was 60.71% in MT10109L group and41.67% in ona-BoNT/A group (Table 3, FIG. 3B). Both PP set and FAS setshowed significant difference in two groups and superiority of MT10109L.

The percentage of responders at rest was assessed at week 4 and 16, andno significant difference was noted between the MT10109L group andona-BoNT/A group at week 4 in both PP set and FAS set. At week 16,percentage of responders at rest in PP set was 50.00% in MT10109L groupand 31.58% in ona-BoNT/A group, showing significant improvement inMT10109L group (p value=0.0482). However, in the FAS set, the percentageof responders did not show significant difference between the MT10109Lgroup (50.00%) and the ona-BoNT/A group (33.33%). (p value: 0.0641)(Table 3).

In photographic assessment, the responder rate at maximum frown at weeks4 by three independent, blinded raters also did not show significantdifference between the

MT10109L group and the ona-BoNT/A group in PP set and FAS set. Theresults are shown in Table 4.

TABLE 4 Responder rate by photo assessment (Weeks 4) MT10109L Ona-BoNT/An (%) n (%) p-value Maximum PP set n 78 81 frown Responder 74 (94.87) 79(97.53) 0.4369‡ non- 4 (5.13) 2 (2.47) responder FAS set n 83 84Responder 79 (95.18) 82 −97.62 0.4431‡ non- 4 (4.82) 2 −2.38 responderResting PP set n 44 47 Responder 25 (56.82) 25 (53.19) 0.7282‡ non- 19(43.18) 22 (46.81) responder FAS set n 48 49 Responder 26 (54.17) 25(51.02) 0.7564‡ non- 22 (45.83) 24 (48.98) responder

Subject's Assessment

Subject's assessment on improvement of glabellar line and satisfactionyielded comparable results for both groups. Peak improvement ratedefined as the proportion of subjects who scored more than +2 points(moderately improved) were assessed at week 4, 10, and 16. In both PPset and FAS set, the peak improvement rate at week 4, 10, and 16 did notshow significant difference between MT10109L and ona-BoNT/A groups.

Subjective-assessment improvement rate was shown 89.74% and 92.59% atweek4, 81.82% and 91.14% at week10, 70.13% and 68.35% at week16,respectively in MT10109L and ona-BoNT/A group of PP set. Satisfactionrate defined as patient showing “very satisfied” or “satisfied” wasassessed at week 4, 10, 16, and this also did not show significantdifference in PP set and FAS set between MT10109L and ona-BoNT/A groups.Satisfaction rate was 78.21% and 71.60% at week4, 74.03% and 68.35% atweek10, 58.44% and 48.10% at week16, respectively in MT10109L andona-BoNT/A group of PP set. The results of PP set are shown in FIG. 4.

Safety

Comparable numbers of treatment-emergent adverse events were reported inthe MT10109L group (21.43%) and the control group (16.67%) (Table 5).There was no adverse drug reaction in MT10109L group. The only adversedrug reaction related to injection was one incident of face edemareported in the control group. No severe adverse drug reaction wasobserved in both groups. No subjects were withdrawn because of adverseevents.

TABLE 5 Adverse events (safety set) MT10109L group Ona-BoNT/A adverseevents (n. %) (N = 84) (N = 84) p-value adverse events 19 (22.62) 15(17.86) 0.4424 treatment-emergent 18 (21.43) 14 (16.67) 0.4319 adverseevents adverse drug reaction facial edema 0 (0.00) 1 (1.19) 1    severeadverse events acute myocardial infarction 0 (0.00) 1 (1.19) 1   peritonitis 0 (0.00) 1 (1.19) 1    rotator cuff syndrome 1 (1.19) 0(0.00) 1   

Serious adverse events happening after the patient receiving the testmedication were reported in 1.19% (1/84 subjects, 1 incidents) of theMT10109L group and 2.38% (2/84 subjects, 2 incidents) of the controlgroup. For serious adverse events, acute myocardial infarction andperitonitis was reported in each patient of ona-BoNT/A group. Thepatient who developed acute myocardial infarction have been treated forhis cardiac problem in the cardiology department, thus, this event doesnot seem to be related to test drug injection. And Rotator cuff syndromewas reported in one patient of MT10109L group after the patient receivedthe test medication.

In terms of the laboratory tests and vital signs, no significantabnormal changes were detected after the test drug administration inMT10109L group and ona-BoNT/A group.

The Efficacy and Safety of Liquid Type Botulinum Toxin Type A

The results presented herein demonstrate that MT10109L is safe andeffective for the treatment of glabellar lines. MT10109L treatmentresulted in significant improvement of glabella frown line severity atboth maximal contraction and rest by live assessment at week 4 and week16. The results of photographic assessment by blinded raters andsubject's assessment in this study indicate that ona-BoNT/A and MT10109Ldid not differ significantly in any variable at any point. The resultalso showed that MT10109L may provide greater improvement thanona-BoNT/A at week 16. The results presented here also suggest thatefficacy of MT10109L persisted longer than ona-BoNT/A. Commerciallyavailable BoNT/A preparations were very diverse and had differentefficacies and safety because of their unique biologic nature (Klein AW, et al., Plast Reconstr Surg., 2008; 121(6):413e-422e). Of these,ona-BoNT/A is the best known type which dominates the botulinum toxinmarket since it was first approved and marketed in the United States in1989 (Yang G H, et al., Dermatologic Surgery, 2013; 39(lpt2):165-170).As a consequence, the majority of the information about handlingbotulinum toxin type A is found with ona-BoNT/A (Trindade De Almeida AR, et al., Dermatologic Surgery, 2011; 37(11):1553-1565), Thus, thisstudy was designed to compare the efficacy and safety of MT10109L andona-BoNT/A.

All of the currently used BoNT/A products recommend that reconstitutionbe performed using variable substances before injection since theproducts are provided as freeze dried powder formulation. These productsalso have discommodity on supply, dilution and storage. For example,Ona-BoNT/A should be stored between 2° C. and 8° C. afterreconstitution, and is to be used within 24 hours of reconstitution(Huang W, et al., J Am Acad Dermatol 2000; 43:249-59).

Although rimabotulinumtoxin b (Myobloc®, Solstice Neurosciences,Louisville, Kent.) has been used as liquid injection form of botulinumtoxin B, it is not widely used as much as BoNT/A and the equivalentdosage of rimabotulinumtoxin b with BoNT/A has not been fully studied(Trindade De Almeida A R, et al., Dermatologic Surgery, 2011;37(11):1553-1565).

MT10109L is the first liquid injection form of BoNT/A. MT10109L containsBoNT/A type macromolecular protein complexes with molecular weights of900 kD, which is similar to ona-BoNT/A (Table 6). MT10109L exhibits asimilar diffusion capacity to that of ona-BoNT/A.

MT10109L is provided as a ready-to-use sterile liquid; no reconstitutionis required and is more convenient to store and reuse compared to mostof BoNT/A. The shelf life of MT10109L is currently estimated at around22 months from the date of manufacture, however, without wishing to bebound by any particular theory, it is expected to be prolonged throughextended stability test. During the period, MT10109L can be reused atanytime without time limit, whereas ona-BoNT/A is recommended to be usedwithin 4 hours to 6 weeks after reconstitution (Carruthers J, et al.,Plast Reconstr Surg., 2004; (Suppl); 11(6)4:2S and Hexsel D M, et al.,Dermatol Surg., 2003; 29:523-9). Because longer stability of MT10109L isvalidated as mentioned elsewhere herein, MT10109L has an advantage overreconstituted ona-BoNT/A in reusing. Moreover, MT10109L is currentlyavailable as small package unit (25 U/vial), which is suitable fortreatment of glabellar lines (Table 6).

TABLE 6 Characteristics of botulinum Toxin Preparations MT10109LOna-BoNT/A Manufacturer Medytox Inc. Allergan, Inc. Commercial namesNeuranox Aqua ® Botox ®, Botox cosmetic ®, Vistabel ®, Vistabex ® Toxinserotype A A Indications glabellar lines Blepharospasm, cervicaldystonia, glabellar lines, hyperhidrosis, chronic migraine Activesubstance Botulinum Toxin Type A Botulinum Toxin Type A Complexmolecular 900 kDa 900 kDa weight U/vial 25 50 Stabilizer/vial methionine(0.125 mg) Human serum albumin (0.5 mg) polysorbate 20 (0.094 mg)Excipients/vial sodium chloride (5.625 mg) sodium chloride (0.45 mg)Formulation liquid vacuum-dried powder Dilution, mL No reconstitutionrequired 1.0-2.5 Storage 2-8° C. 2-8° C. or < −5° C. Storage afterwithout limitation/2-8° C. 24 hours/2-8° C. dilution/temperature

The results presented herein demonstrate that the efficacy of MT10109Lpersisted longer than ona-BoNT/A. The therapeutic effect of BoNT/A forthe glabellar line improvement could be detected within 4 weeks afterinjection and gradually decrease between 3-6 months. The fact that alonger maintaining period of the glabellar line improvement associatedwith the use of MT10109L provides a great advantage over other types ofBoNT/A. Results from a meta-analysis by Glogau R, et al. showedtreatment of glabellar line with 20 unit of ona-BoNT/A sustainedclinical effect at week 16 in more than 50% of responders (Glogau R, etal., Dermatol Surg., 2012; 38(11):1794-803). However, in this study,responder rate of ona-BoNT/A was lower than that of previous reports.Thus, large studies, with enrollment of patients from several medicalcenters and with longer follow-up periods, are needed to confirm thisresult.

Whereas currently used BoNT/A contain albumin or gelatin forstabilization, MT10109L eliminated albumin but also animal derivedmaterials in component of the entire manufacturing process. Therefore,MT10109L minimized risk of infectious diseases which includedtransmissible spongiform encephalopathy. No severe adverse drugreactions with either toxin were observed in the present study.Therefore, MT10109L is as safe as ona-BoNT/A.

The results presented herein demonstrate that MT10109L is not inferiorto ona-BoNT/A in the improvement of glabellar lines and is relativelysimilar in safety, which therefore can be judged to be used in thetreatment of the relevant symptom. With its longer maintaining period ofthe glabellar line improvement, convenience without the additionaldilution step, easy storage and re-usage, and animal derivedprotein-free constituents, MT10109L is a desirable substitute for theconventional powder formulation of BoNT/A.

Example 2 Randomized, Double-blind, Multi-center, Phase II, OptimalDose-finding Study to Determine Safety and Efficacy of MT10109 v. BOTOXin Subjects with Moderate to Severe Glabellar Lines

The results presented herein compare the safety and efficacy oflyophilized formulation of MT10109 and BOTOX® in subjects with moderateto severe glabellar lines. It is demonstrated that the response atmaximum frown was sustained in the MT10109 20U group for up to 120 days.

The experiments described herein compared dosing of MT10109 at 10 U, 20U and 30 U to BOTOX® dosed at 20U. The efficacy of MT10109 was primarilyassessed at Day 30 (Visit 4; ±7 days) and the comparison of interest wasthe comparison between the responder rates for MT10109 20 U and BOTOX®20 U. A responder was defined as a glabellar line severity rating ofnone (0) or mild (1) at maximum frown or at rest at Day 30, depending onthe analysis.

The time points of Day 14 (Visit 3), Day 30 (Visit 4), Day 60 (Visit 5),Day 90 (Visit 6) and Day 120 (Visit 7) were allowed 7 days either sideof the visit day and so Day 30, for example, may not be exactly 30 daysafter study treatment administration. As presented herein, time pointshave been labelled by day rather than visit. All analyses of efficacywere performed using the Full Analysis Set (FAS) and were repeated usingthe Per-protocol (PP) Set as a supportive analysis.

Primary Efficacy Parameter: Investigator's Rating of Glabellar LineSeverity at Maximum Frown at Day 30 by Live Assessment

The investigator's live assessment of glabellar line severity at maximumfrown at Day 30 is summarized for the FAS in Table 7. A Mantel-Haenszelchi-square test was used to compare treatment groups and is in diagramform in FIG. 5. Results for the

PP Set are summarized in Table 8.

TABLE 7 Investigator's Live Assessment of Glabellar Line Severity atMaximum Frown at Day 30, Full Analysis Set MT10109 10U MT10109 20UMT10109 30U BOTOX 20U Live Assessment of N = 31 N = 28 N = 26 N = 29Glabellar Lines n (%) n (%) n (%) n (%) Day 30, at n = 30 n = 26 n = 25n = 26 maximum frown Responders 18 (60.0) 18 (69.2) 22 (88.0) 19 (73.1)Non-responders 12 (40.0)  8 (30.8)  3 (12.0)  7 (26.9) Abbreviations: N= number of subjects in analysis set; n = number of subjects with data.Note: Subjects included in the analysis had to have a baseline glabellarline severity rating at maximum frown of moderate (2) or severe (3). Aresponder was defined as having a severity rating of none (0) or mild(1) at the corresponding post-baseline visit. Percentages are based onthe number of subjects with an assessment at the relevant visit.

For the FAS, based on the investigator's live assessment of subjects'glabellar line severity at maximum frown at Day 30, the proportion ofresponders (a severity rating of none [0] or mild [1]) in the MT10109 20U group was similar to that in the BOTOX® 20 U group (Table 7).

The proportion of responders in the MT10109 20 U group was 69.2% (18 of26 subjects) and in the BOTOX® 20 U group was 73.1% (19 of 26 subjects).

There was no statistically significant difference between the proportionof responders in the MT10109 20 U and BOTOX® 20 U groups (−3.2 [95% CI:−28.3 to 21.8]; p-value 0.760) (FIG. 5).

The proportion of responders in the MT10109 10 U group was smallercompared with the BOTOX® 20 U group and the proportion of responders inthe MT10109 30 U group was greater compared with BOTOX® 20 U (Table 7).

The proportion of responders in the MT10109 10 U group was 60.0% (18 of30 subjects) and in the MT10109 30 U group was 88.0% (22 of 25 subjects)and in the BOTOX® 20 U group was 73.1% (19 of 26 subjects).

There was no statistically significant difference between the proportionof responders in the MT10109 10 U and BOTOX® 20 U groups (−9.7 [95% CI:−34.6 to 15.2]; p-value 0.448), or between the MT10109 30 U and BOTOX®20 U groups (17.6 [95% CI: −4.1 to 39.3]; p-value 0.117) (FIG. 5).

There was no statistically significant difference between the proportionof responders in the MT10109 10 U and MT10109 20 U groups (−6.5 [95% CI:−31.4 to 18.4]; p-value 0.614), or between the proportion of respondersin the MT10109 30 U and MT10109 20 U groups (20.8 [95% CI: −0.9 to42.5]; p-value 0.066). The proportion of responders in the MT10109 10 Ugroup was smaller than that of the MT10109 30 U group and the differencewas statistically significant (−27.3 [95% CI: −48.8 to −5.8]; p-value0.020).

The investigator's live assessment of glabellar line severity at maximumfrown at Day 30 is summarized for the PP Set in Table 8. AMantel-Haenszel chi-square test was used to compare treatment groups.

TABLE 8 Investigator's Live Assessment Rating of Glabellar Line Severityat Maximum Frown at Day 30, Per-protocol Set MT10109 10U MT10109 20UMT10109 30U BOTOX 20U Live Assessment of N = 30 N = 26 N = 23 N = 26Glabellar Lines n (%) n (%) n (%) n (%) Day 30, at maximum frown n = 30n = 26 n = 23 n = 26 Responders 18 (60.0) 18 (69.2) 20 (87.0) 19 (73.1)Non-responders 12 (40.0)  8 (30.8)  3 (13.0)  7 (26.9) Abbreviations: N= number of subjects in analysis set; n = number of subjects with data.Note: Subjects included in the analysis had to have a baseline glabellarline severity rating of moderate (2) or severe (3). A responder wasdefined as having a severity rating of none (0) or mild (1) at thecorresponding post-baseline visit. Percentages are based on the numberof subjects with an assessment at the relevant visit.

The results in the PP Set supported those in the FAS.

The proportion of responders in the MT10109 20 U group was 69.2% (18 of26 subjects) and in the BOTOX® 20 U group was 73.1% (19 of 26 subjects)(Table 8).

The difference between the proportion of responders in the MT10109 20 Uand BOTOX® 20 U groups was −3.8 (95% CI: −32.8 to 25.1; p-value 0.762)which was not statistically significant.

The proportion of responders in the PP Set in the MT10109 10 U group wassmaller compared with the BOTOX® 20 U group and the proportion ofresponders in the MT10109 30 U group was greater compared with BOTOX® 20U:

The proportion of responders in the MT10109 10 U group was 60.0% (18 of30 subjects) and in the MT10109 30 U group was 87.0% (20 of 23 subjects)and in the BOTOX® 20 U group was 73.1% (19 of 26 subjects) (Table 8).

There was no statistically significant difference between the proportionof responders in the MT10109 10 U and BOTOX® 20 U groups (−13.1 [95% CI:−41.6 to 15.4]; p-value 0.307), or between the MT10109 30 U and BOTOX®20 U groups (13.9 [95% CI: −12.6 to 40.3]; p-value 0.234).

Secondary Efficacy Parameter: Investigator's Rating of Glabellar LineSeverity at Maximum Frown and at Rest up to Day 120 by Live Assessment

The investigator's live assessment of glabellar line severity at maximumfrown and rest at other visits is summarized for the FAS in Table 9, andin diagram form in FIG. 6. A Mantel-Haenszel chi square test was used tocompare treatment groups.

TABLE 9 Investigator's Live Assessment Rating of Glabellar Line Severityat Other Visits, Full Analysis Set MT10109 10U MT10109 20U MT10109 30UBOTOX 20U Live Assessment of N = 31 N = 28 N = 26 N = 29 Glabellar Linesn (%) n (%) n (%) n (%) Day 14, at maximum frown n = 31 n = 28 n = 25 n= 28 Responders 18 (58.1) 20 (71.4) 22 (88.0) 22 (78.6) Non-responders13 (41.9)  8 (28.6)  3 (12.0)  6 (21.4) Day 14, at rest n = 13 n = 12 n= 14 n = 11 Responders  4 (30.8)  3 (25.0)  8 (57.1)  7 (63.6)Non-responders  9 (69.2)  9 (75.0)  6 (42.9)  4 (36.4) Day 30, at rest n= 12 n = 10 n = 13 n = 9 Responders  4 (33.3)  1(10.0)  6 (46.2)  5(55.6) Non-responders  8 (66.7)  9 (90.0)  7 (53.8)  4 (44.4) Day 60, atmaximum frown n = 30 n = 23 n = 25 n = 25 Responders 15 (50.0) 15 (65.2)17 (68.0) 17 (68.0) Non-responders 15 (50.0)  8 (34.8)  8 (32.0)  8(32.0) Day 60, at rest n = 12 n = 10 n = 13 n = 10 Responders  4 (33.3)1(10.0)  7 (53.8)  4 (40.0) Non-responders  8 (66.7)  9 (90.0)  6 (46.2) 6 (60.0) Day 120, at maximum frown n = 28 n = 23 n = 25 n = 26Responders  9 (32.1) 12 (52.2)  7 (28.0)  6 (23.1) Non-responders 19(67.9) 11 (47.8) 18 (72.0) 20 (76.9) Day 120, at rest n = 10 n = 10 n =13 n = 10 Responders  1(10.0)  1(10.0)  5 (38.5)  3 (30.0)Non-responders  9 (90.0)  9 (90.0)  8 (61.5)  7 (70.0) Abbreviations: N= number of subjects in analysis set; n = number of eligible subjectswith data. Note: Subjects included in the analysis had to have abaseline glabellar line severity rating of moderate (2) or severe (3). Aresponder was defined as having a severity rating of none (0) or mild(1) at the corresponding post-baseline visit. Percentages are based onthe number of eligible subjects with an assessment at the relevantvisit.

The proportion of responders at maximum frown decreased in all treatmentgroups from Day 14 to Day 120 (based on the investigator's liveassessment). The proportions at Day 120 were greater in the MT10109 20 Ugroup compared with BOTOX® 20 U and the other MT10109 groups (FIG. 6).

The proportion of responders at maximum frown in the MT10109 20 U groupat Day 60, and at Day 120 was 65.2% (15 of 23 subjects) and 52.2% (12 of23 subjects), respectively, and in the BOTOX® 20 U group was 68.0% (17of 25 subjects) and 23.1% (6 of 26 subjects), respectively (Table 9).

The difference between the proportion of responders in the MT10109 20 Uand BOTOX® 20 U groups at maximum frown at Day 60 was −4.3 (95% CI:−30.7 to 22.1; p-value 0.714) and at Day 120 was 24.0 (−0.7 to 48.7;p-value 0.058).

In the other MT10109 groups, the proportions of responders at maximumfrown at Day 60 were smaller in the MT10109 10 U group (50.0%; 15 of 30subjects) and similar in the MT10109 30 U group (68.0%; 17 of 25subjects) compared with the BOTOX® 20 U group (68.0%; 17 of 25subjects). At Day 120 proportions were greater in the MT10109 10 U(32.1%; 9 of 28 subjects) and MT10109 30 U (28.0%; 7 of 25 subjects)groups than in the BOTOX® 20 U group (23.1%;6 of 26 subjects) (Table 9).There was no statistically significant difference in the proportion ofresponders at maximum frown between the MT10109 10 U and BOTOX® 20 Ugroups or between the MT10109 30 U and BOTOX® 20 U groups at any timepoint. There was no statistically significant difference in theproportion of responders at maximum frown between the MT10109 10 U andMT10109 20 U groups, or between the MT10109 30 U and MT10109 20 U groupsat any time point. The proportion of responders in the MT10109 10 Ugroup was generally smaller than in the MT10109 30 U group and thedifference was statistically significant at Day 14 (−30.4 [95% CI: −55.6to −5.2]; p-value 0.012).

In the analyses of the investigator's live assessment of glabellar lineseverity at rest, the number of subjects who were eligible for inclusionin the analysis (i.e., baseline glabellar line severity of eithermoderate [2] or severe [3]) was small. Consequently, comparisons betweentreatment groups and any resulting p-values should be treated withcaution.

The proportion of responders at rest decreased in all treatment groupsfrom Day 14 to Day 120 (based on the investigator's live assessment)(FIG. 9 center left). The proportions were smaller in the MT10109 20 Ugroup compared with the BOTOX® 20 U group at every time point.

The proportion of responders at rest in the MT10109 20 U group at Day 14and at Day 120 was 25.0% (3 of 12 subjects) and 10.0% (1 of 10subjects), respectively, and in the BOTOX® 20 U group was 63.6% (7 of 11subjects) and 30.0% (3 of 10 subjects), respectively (Table 9).

Proportions of responders at rest at Day 120 were smaller in the MT1010910 U (10.0%; 1 of 10 subjects) and greater in the MT10109 30 U (38.5%; 5of 13 subjects) groups than in the BOTOX® 20 U group (30.0%; 3 of 10subjects) (Table 9).

Secondary Efficacy Parameter: Subject's Assessment of Glabellar LineImprovement and Satisfaction with Effect of Treatment

The subject's self-assessment of glabellar line improvement issummarized in Table 10 and in diagram form in FIG. 7. The subject'sself-assessment of satisfaction with the effect of treatment issummarized in Table 11 and in diagram form in FIG. 8. A Mantel-Haenszelchi square test was used to compare treatment.

TABLE 10 Subject's Self-assessment of Glabellar Line Improvement, FullAnalysis Set MT10109 MT10109 MT10109 BOTOX 10U 20U 30U 20USelf-assessment N = 31 N = 28 N = 26 N = 29 Improvement n (%) n (%) n(%) n (%) Day 14 n = 31 n = 28 n = 25 n = 28 Responders 18 (58.1) 21(75.0) 22 (88.0) 23 (82.1) Non-responders 13 (41.9)  7 (25.0)  3 (12.0) 5 (17.9) Day 30 n = 30 n = 26 n = 25 n = 26 Responders 16 (53.3) 19(73.1) 19 (76.0) 18 (69.2) Non-responders 14 (46.7)  7 (26.9)  6 (24.0) 8 (30.8) Day 60 n = 30 n = 23 n = 25 n = 25 Responders 13 (43.3) 15(65.2) 17 (68.0) 13 (52.0) Non-responders 17 (56.7)  8 (34.8)  8 (32.0)12 (48.0) Day 90 n = 30 n = 25 n = 25 n = 26 Responders 6 (20.0) 14(56.0) 10 (40.0)  9 (34.6) Non-responders 24 (80.0) 11 (44.0) 15 (60.0)17 (65.4) Day 120 n = 28 n = 23 n = 25 n = 26 Responders  8 (28.6) 10(43.5)  7 (28.0)  8 (30.8) Non-responders 20 (71.4) 13 (56.5) 18 (72.0)18 (69.2) Abbreviations: N = number of subjects in analysis set; n =number of subjects with data. Note: Subjects included in the analysishad to have a baseline glabellar line severity rating of moderate (2) orsevere (3). A responder was defined as having a score of at least +2(moderate improvement, about 50%) on the 9-point scale. Percentages arebased on the number of subjects with an assessment at the relevantvisit.

In the subject's self-assessment of glabellar line improvement, theproportions of responders (a score of at least +2 [moderate improvement,about 50%] on the 9-point scale) at Day 30 were similar across theMT10109 20 U and BOTOX® 20 U groups (FAS), which reflected the liveassessment ratings. The proportions were also similar across the MT1010930 U and BOTOX® 20 U groups. At Day 60 the proportions were greater inthe MT10109 20 U and MT10109 30 U groups than the BOTOX® 20 U group andat Day 120, the proportions remained greater in the MT10109 20 U groupcompared with the other treatment groups (FIG. 7).

The proportion of responders in the MT10109 20 U group at Day 30, Day 60and Day 120 was 73.1% (19 of 26 subjects), 65.2% (15 of 23 subjects) and43.5% (10 of 23 subjects), respectively, and in the BOTOX® 20 U groupwas 69.2% (18 of 26 subjects), 52.0% (13 of 25 subjects) and 30.8% (8 of26 subjects), respectively (Table 10). There was no statisticallysignificant difference between the proportion of responders in theMT10109 20 U and BOTOX® 20 U groups at any time point.

In the other MT10109 groups, at Day 30 and Day 60, proportions ofresponders were smaller in the MT10109 10 U group and greater in theMT10109 30 U group compared with the BOTOX® 20 U group (Table 10). AtDay 120, proportions were similar across the three treatment groups(MT10109 10 U, 28.6% [8 of 28 subjects]; MT10109 30 U, 28.0% [7 of 25subjects]; BOTOX® 20 U, 30.8% [8 of 26 subjects]). The difference in theproportion of responders between MT10109 10 U and BOTOX® 20 U at Day 14was statistically significant (−23.6 [95% CI: −46.5 to −0.6]; p-value0.049). No statistically significant difference between the proportionof responders in the MT10109 30 U and BOTOX® 20 U groups was observed atany time point. There was no statistically significant difference in theproportion of responders in the subject's assessment of glabellar lineimprovement between the MT10109 30 U and MT10109 20 U groups. Theproportion of responders in the MT10109 10 U group was smaller than inthe other two groups at each time point. The difference wasstatistically significant between the MT10109 10 U and MT10109 30 Ugroups at Day 14 (−30.4 [95% CI: −52.0 to −8.7]; p-value 0.011) andbetween the MT10109 10 U and MT10109 20 U groups at Day 90 (−34.7 [95%CI: −58.8 to −10.7]; p-value 0.007).

TABLE 11 Subject's Self-assessment of Satisfaction with the Effect ofthe Treatment, Full Analysis Set MT10109 MT10109 MT10109 BOTOX 10U 20U30U 20U Self-assessment N = 31 N = 28 N = 26 N = 29 Satisfaction n (%) n(%) n (%) n (%) Day 14 n = 31 n = 28 n = 25 n = 28 Responders 16 (51.6)20 (71.4) 19 (76.0) 17 (60.7) Non-responders 15 (48.4)  8 (28.6)  6(24.0) 11 (39.3) Day 30 n = 30 n = 26 n = 25 n = 26 Responders 13 (43.3)17 (65.4) 18 (72.0) 16 (61.5) Non-responders 17 (56.7)  9 (34.6)  7(28.0) 10 (38.5) Day 60 n = 30 n = 23 n = 25 n = 25 Responders 14 (46.7)14 (60.9) 13 (52.0) 11 (44.0) Non-responders 16 (53.3)  9 (39.1) 12(48.0) 14 (56.0) Day 90 n = 30 n = 25 n = 25 n = 26 Responders 10 (33.3)14 (56.0) 15 (60.0)  9 (34.6) Non-responders 20 (66.7) 11 (44.0) 10(40.0) 17 (65.4) Day 120 n = 28 n = 23 n = 25 n = 26 Responders 11(39.3) 13 (56.5) 11 (44.0) 12 (46.2) Non-responders 17 (60.7) 10 (43.5)14 (56.0) 14 (53.8) Abbreviations: N = number of subjects in analysisset; n = number of subjects with data. Note: Subjects included in theanalysis had to have a baseline glabellar line severity rating ofmoderate (2) or severe (3). A responder was defined as having a score ofat least 6 (satisfied) on the 7-point scale. Percentages are based onthe number of subjects with an assessment at the relevant visit.

In the subject's self-assessment of satisfaction with the effect oftreatment, a responder was defined as having a score of at least 6(satisfied) on the 7-point scale.

As seen in the self-assessment of improvement, the proportion ofresponders in the assessment of satisfaction at Day 30 were similar inboth the MT10109 20 U and BOTOX® 20 U groups, which reflected the liveassessment ratings. At Day 120, the responder rate was greater in theMT10109 20 U group than any other treatment group (FIG. 8). Satisfactionappeared consistent in the MT10109 20 U group but a pattern was hard todiscern in the BOTOX® 20 U and other MT10109 groups.

The proportion of responders in the MT10109 20 U group at Day 30, Day 60and Day 120 was 65.4% (17 of 26 subjects), 60.9% (14 of 23 subjects) and56.5% (13 of 23 subjects), respectively, and in the BOTOX® 20 U groupwas 61.5% (16 of 26 subjects), 44.0% (11 of 25 subjects) and 46.2% (12of 26 subjects), respectively (Table 11). There was no statisticallysignificant difference in the proportion of responders between theMT10109 20 U and BOTOX® 20 U groups at any time point.

In the other MT10109 groups, at Day 30, proportions of responders weresmaller in the MT10109 10 U group and greater in the MT10109 30 U groupcompared with the BOTOX® 20 U group (Table 11). At Day 60, proportionswere greater in both MT10109 groups compared with BOTOX® 20 U. At Day120, proportions were smaller in the MT10109 10 U (39.3% [11 of 28subjects]) and MT10109 30 U (44.0% [11 of 25 subjects]) treatment groupscompared with the BOTOX® 20 U group (46.2% [12 of 26 subjects]). Nostatistically significant difference in the proportion of respondersbetween the MT10109 10 U and BOTOX® 20 U groups or the MT10109 30 U andBOTOX® 20 U groups was observed at any time point.

There was no statistically significant difference in the proportion ofresponders in the subject's assessment of satisfaction with the effectof treatment between the MT10109 10 U and MT10109 20 U groups, orbetween the MT10109 30 U and MT10109 20 U groups. The proportion ofresponders in the MT10109 10 U group was smaller than in the MT10109 30U group at each time point and the difference was statisticallysignificant at Day 30 (−27.3 [95% CI: −52.8 to −1.9]; p-value 0.040).

Efficacy Results (FAS)—Improved Sustained Efficacy of MT10109

MT10109 at a single dose of 20 U demonstrated a similar effect to thatof BOTOX® 20 U in reducing subjects' glabellar lines.

After the investigator's live assessment of glabellar line severity atmaximum frown at Day 30, the proportion of responders in the MT10109 20U group was 69.2% and in the BOTOX® 20 U group was 73.1%. The differencebetween the proportion of responders in the two treatment groups was−3.2 (95% CI: −28.3 to 21.8) which was not statistically significant(p-value 0.760).

In the subject's self-assessment of glabellar line improvement andsatisfaction with the effect of treatment, proportions of responderswere generally greater in the MT10109 20 U and MT10109 30 U groupscompared with BOTOX® 20 U. At Day 30, responder rates in the MT10109 20U and BOTOX® 20 U groups were similar, reflecting the live assessmentratings (MT10109 20 U group, 73.1% for improvement and 65.4% forsatisfaction with treatment effect; BOTOX® 20 U group, 69.2% and 61.5%,respectively).

The response at maximum frown was sustained in the MT10109 20 U group toDay 120. The proportion of responders at Day 120, according to theinvestigator's live assessment, was 52.2% in the MT10109 20 U groupcompared with 23.1% in the BOTOX® 20 U group. At most time points,proportions of responders at maximum frown were greater in the MT1010930 U group and smaller in the MT10109 10 U group compared with theBOTOX® 20 U group.

Fewer subjects were considered responders by the independent reviewersbased on the photographic assessment; however, the proportions ofresponders (using the mean score) at maximum frown at Day 30 and Day 60were similar in the MT10109 20 U (48.1% and 40.7%, respectively) andBOTOX® 20 U treatment groups (51.7% and 41.4%, respectively), whichreflected the investigator's live assessment.

In both the investigator's live assessment and independent reviewers'photographic assessment of glabellar lines severity at rest, theproportions of responders were small across all treatment groups. Atmost time points, the responder rates were smaller in the MT10109 20 Ugroup compared with the BOTOX® 20 U group.

Using a logistic regression at Day 30 and at maximum frown, thedose-response curve showed no relationship between dose and response.

The data from the experiments is further illustrated in FIG. 9 and FIG.10. FIG. 9 presents a series of graphs depicting investigator assessmentat maximum frown and at rest, independent reader assessment at maximumfrown and at rest, and the subjects' assessment and satisfaction. FIG.10 illustrates the data as generated collectively, and broken into thetwo different treatment sites.

In summary, the data presented herein demonstrate that lyophilizedMT10109 dosed at 20U demonstrates similarity to BOTOX® at early timepoints (e.g. day 30). Further, it is demonstrated that MT10109 dosed at20U displays an increased sustained effect compared to BOTOX®, as theresponse of treatment was seen to be increased in the MT10109 20U groupcompared to BOTOX® 20U group at 120 days post treatment.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

We claim:
 1. A method to reduce frequency of treatment with botulinumtoxin, comprising: providing an animal-protein free botulinum toxincomposition for local administration by injection at a treatment site,wherein administration of a first therapeutically effective amount isseparated by a time interval from administration of a secondtherapeutically effective amount, said time interval being greater thana time interval between first and second treatment of a therapeuticallyeffective amount of an animal-protein containing botulinum toxincomposition administered by injection at the treatment site.
 2. Themethod of claim 1, wherein during said time interval betweenadministration of the first and second therapeutically effective amountsof the animal-protein free botulinum toxin composition a symptom in apatient is alleviated.
 3. The method of claim 1, wherein providingcomprising providing a liquid, animal-protein free botulinum toxincomposition.
 4. The method of claim 3, wherein the liquid compositioncomprises a polysorbate and methionine.
 5. The method of claim 1,wherein providing comprising providing a lyophilized, animal-proteinfree botulinum toxin composition.
 6. The method of claim 3, wherein theliquid composition comprises a polysorbate, methionine and a compoundselected from the group consisting of a sugar, a sugar alcohol and anionic compound.
 7. The method of claim 1, wherein the time interval isgreater than about 16 weeks.
 8. A method to increase efficacy ofbotulinum toxin, comprising: providing an animal-protein free botulinumtoxin composition for local administration by injection at a treatmentsite, wherein administration of a first therapeutically effective amountis separated by a time interval from administration of a secondtherapeutically effective amount, said time interval being greater thana time interval between first and second treatment of a therapeuticallyeffective amount of an animal-protein containing botulinum toxincomposition administered by injection at the treatment site.
 9. A methodof botulinum toxin treatment, comprising: providing an animal-proteinfree botulinum toxin composition for local administration at a treatmentsite, the composition comprising botulinum toxin, a polysorbate andmethionine, wherein administration of a therapeutically effective amountof the composition alleviates a symptom in a patient for a period oftime greater than that provided by administration of a therapeuticallyeffective amount of an animal-protein containing botulinum toxincomposition locally administered at the treatment site.